Evolution of virulence in heterogeneous host communities under multiple trade-offs

Many pathogens and parasites are transmitted through hosts that differ in species, sex, genotype, or immune status. In addition, virulence (here defined as disease-induced mortality) and transmission can vary during the infectious period within hosts of different state. Most models of virulence evolution assume that transmission and virulence are constant over the infectious period and that the host population is homogenous. Here, we examine a multispecies susceptible-infected-recovered (SIR) model where transmission occurs within and between species, and transmission and virulence varied during the infectious period. This allows us to understand virulence evolution in a broader range of situations that characterize many emerging diseases. Because emerging pathogens are by definition new to their host populations, they should be expected to rapidly adapt after emergence. We illustrate these evolutionary effects using the framework of adaptive dynamics to examine how virulence evolves after emergence in response to the relative strength of selection on pathogen fitness and mutational variance for virulence. We illustrate the role of evolution by simulating adaptive walks to an evolutionarily stable virulence. We found that the magnitude of between-species transmission and the relative timing of transmission and mortality across species were of primary importance for determining the evolutionarily stable virulence.     

On the evolution of virulence and the relationship between various measures of mortality

Smallpox causes roughly 20% mortality whereas chickenpox causes less than 0.1%. Most 'verbal' (i.e. non-mathematical) discussions using a mortality definition of virulence would therefore label smallpox as more virulent. Indeed, the virulence of many diseases is measured using such case mortalities, chi, or related measures such as expected host lifespan, T, or lethal dose, LD(x). But chi, T and LD(x) are only indirectly related to parasite-induced instantaneous mortality rate, alpha, which is the mortality measure used in much of the theory developed to explain virulence evolution. Here I point out that relatively deadly pathogens can actually have lower values of alpha than benign pathogens, demonstrating that alpha does not, by itself, reflect the extent to which a parasite causes host mortality. I present mathematical relationships between alpha and chi, T and LD(x), and use these to demonstrate that predictions about virulence evolution can be qualitatively altered depending upon which measure is used as the definition of virulence. Two simple examples are presented to illustrate this point, one of which demonstrates that the well-cited prediction that virulence should evolve to be higher when disease-independent host mortality increases need not hold. This prediction has been made in terms of parasite-induced instantaneous mortality, alpha, but if virulence is measured using case mortality (or T or LD(x)) then this prediction can easily be reversed. Theoretical and empirical researchers must use compatible mortality measures before a productive exchange between the two can take place, and it is suggested that case mortality (or lethal dose) is best suited as a single (mortality) measure of parasite virulence.     

Interactions between host sex and age of exposure modify the virulence–transmission trade‐off

The patterns of immunity conferred by host sex or age represent two sources of host heterogeneity that can potentially shape the evolutionary trajectory of disease. With each host sex or age encountered, a pathogen's optimal exploitative strategy may change, leading to considerable variation in expression of pathogen transmission and virulence. To date, these host characteristics have been studied in the context of host fitness alone, overlooking the effects of host sex and age on the fundamental virulence–transmission trade‐off faced by pathogens. Here, we explicitly address the interaction of these characteristics and find that host sex and age at exposure to a pathogen affect age‐specific patterns of mortality and the balance between pathogen transmission and virulence. When infecting age‐structured male and female Daphnia magna with different genotypes of Pasteuria ramosa, we found that infection increased mortality rates across all age classes for females, whereas mortality only increased in the earliest age class for males. Female hosts allowed a variety of trade‐offs between transmission and virulence to arise with each age and pathogen genotype. In contrast, this variation was dampened in males, with pathogens exhibiting declines in both virulence and transmission with increasing host age. Our results suggest that differences in exploitation potential of males and females to a pathogen can interact with host age to allow different virulence strategies to coexist, and illustrate the potential for these widespread sources of host heterogeneity to direct the evolution of disease in natural populations.     

Pathogen genomic surveillance elucidates the origins,transmission and evolution of emerging viral agents in China

In the past twenty years, numerous novel zoonotic viral agents with pandemic potential have emerged in China, such as the severe acute respiratory syndrome (SARS) coronavirus and, more recently, the avian-origin influenza A/H7N9 virus, which have caused outbreaks among humans with high morbidity and mortality. In addition, several emerging and re-emerging viral pathogens have also been imported into China from travelers, e.g. the Middle East respiratory syndrome (MERS) coronavirus and Zika virus (ZIKV). Herein, we review these emerging viral pathogens in China and focus on how surveillance by pathogen genomics has been employed to discover and annotate novel pathogenic agents, identify natural reservoirs, monitor the transmission events and delineate their evolution and adaption to the human host. We also highlight the application of genomic sequencing in the recent Ebola epidemics in Western Africa. In summary, genomic sequencing has become a standard research tool in the field of emerging infectious diseases which has been proven invaluable in containing these viral infections and reducing burden of disease in humans and animals. Genomic surveillance of pathogenic agents will serve as a key epidemiological and research tool in the modern era of precision infectious diseases and in the future studies of virosphere.     

A Multiplex PCR/LDR Assay for the Simultaneous Identification of Category A Infectious Pathogens: Agents of Viral Hemorrhagic Fever and Variola Virus

CDC designated category A infectious agents pose a major risk to national security and require special action for public health preparedness. They include viruses that cause viral hemorrhagic fever (VHF) syndrome as well as variola virus, the agent of smallpox. VHF is characterized by hemorrhage and fever with multi-organ failure leading to high morbidity and mortality. Smallpox, a prior scourge, has been eradicated for decades, making it a particularly serious threat if released nefariously in the essentially non-immune world population. Early detection of the causative agents, and the ability to distinguish them from other pathogens, is essential to contain outbreaks, implement proper control measures, and prevent morbidity and mortality. We have developed a multiplex detection assay that uses several species-specific PCR primers to generate amplicons from multiple pathogens; these are then targeted in a ligase detection reaction (LDR). The resultant fluorescently-labeled ligation products are detected on a universal array enabling simultaneous identification of the pathogens. The assay was evaluated on 32 different isolates associated with VHF (ebolavirus, marburgvirus, Crimean Congo hemorrhagic fever virus, Lassa fever virus, Rift Valley fever virus, Dengue virus, and Yellow fever virus) as well as variola virus and vaccinia virus (the agent of smallpox and its vaccine strain, respectively). The assay was able to detect all viruses tested, including 8 sequences representative of different variola virus strains from the CDC repository. It does not cross react with other emerging zoonoses such as monkeypox virus or cowpox virus, or six flaviviruses tested (St. Louis encephalitis virus, Murray Valley encephalitis virus, Powassan virus, Tick-borne encephalitis virus, West Nile virus and Japanese encephalitis virus).     

Emerging concepts in the pathogenesis of the Streptococcus pneumoniae: From nasopharyngeal colonizer to intracellular pathogen

Streptococcus pneumoniae (the pneumococcus) is a human respiratory tract pathogen and a major cause of morbidity and mortality globally. Although the pneumococcus is a commensal bacterium that colonizes the nasopharynx, it also causes lethal diseases such as meningitis, sepsis, and pneumonia, especially in immunocompromised patients, in the elderly, and in young children. Due to the acquisition of antibiotic resistance and the emergence of nonvaccine serotypes, the pneumococcus has been classified as one of the priority pathogens for which new antibacterials are urgently required by the World Health Organization, 2017. Understanding molecular mechanisms behind the pathogenesis of pneumococcal infections and bacterial interactions within the host is crucial to developing novel therapeutics. Previously considered to be an extracellular pathogen, it is becoming evident that pneumococci may also occasionally establish intracellular niches within the body to escape immune surveillance and spread within the host. Intracellular survival within host cells also enables pneumococci to resist many antibiotics. Within the host cell, the bacteria exist in unique vacuoles, thereby avoiding degradation by the acidic lysosomes, and modulate the expression of its virulence genes to adapt to the intracellular environment. To invade and survive intracellularly, the pneumococcus utilizes a combination of virulence factors such as pneumolysin (PLY), pneumococcal surface protein A (PspA), pneumococcal adhesion and virulence protein B (PavB), the pilus‐1 adhesin RrgA, pyruvate oxidase (SpxB), and metalloprotease (ZmpB). In this review, we discuss recent findings showing the intracellular persistence of Streptococcus pneumoniae and its underlying mechanisms.     

Vector-Borne Pathogen and Host Evolution in a Structured Immuno-Epidemiological System

Vector-borne disease transmission is a common dissemination mode used by many pathogens to spread in a host population. Similar to directly transmitted diseases, the within-host interaction of a vector-borne pathogen and a host’s immune system influences the pathogen’s transmission potential between hosts via vectors. Yet there are few theoretical studies on virulence–transmission trade-offs and evolution in vector-borne pathogen–host systems. Here, we consider an immuno-epidemiological model that links the within-host dynamics to between-host circulation of a vector-borne disease. On the immunological scale, the model mimics antibody-pathogen dynamics for arbovirus diseases, such as Rift Valley fever and West Nile virus. The within-host dynamics govern transmission and host mortality and recovery in an age-since-infection structured host-vector-borne pathogen epidemic model. By considering multiple pathogen strains and multiple competing host populations differing in their within-host replication rate and immune response parameters, respectively, we derive evolutionary optimization principles for both pathogen and host. Invasion analysis shows that the \({\mathcal {R}}_0\) maximization principle holds for the vector-borne pathogen. For the host, we prove that evolution favors minimizing case fatality ratio (CFR). These results are utilized to compute host and pathogen evolutionary trajectories and to determine how model parameters affect evolution outcomes. We find that increasing the vector inoculum size increases the pathogen \({\mathcal {R}}_0\), but can either increase or decrease the pathogen virulence (the host CFR), suggesting that vector inoculum size can contribute to virulence of vector-borne diseases in distinct ways.     

The evolution of virulence in pathogens with frequency-dependent transmission

Frequency-dependent transmission is an important feature of diseases that are sexually transmitted or transmitted by a vector that actively searches for hosts. Here I describe the evolution of virulence in pathogens that have frequency-dependent transmission. I consider two components of virulence--an increase in host mortality due to infection, as is classically described, and a decrease in host fecundity due to infection, because frequency dependence is common among diseases that fully or partially sterilize their hosts. Theoretical predictions pertaining to host-pathogen numerical dynamics can be quite different between pathogens with frequency-dependent transmission and those with density-dependent transmission. In contrast, this study suggests that the principles governing the evolution of virulence that have been established in the context of density-dependent pathogens may also apply (qualitatively) to frequency-dependent pathogens. I examine the evolutionary trajectories of the mortality and sterility components of virulence as well as the role of spatial population structure in the evolution of the sterility component of virulence.     

冠状病毒S蛋白及其受体的结构和功能

冠状病毒是有包膜的单股正链RNA病毒。作为人和动物的重要致病原,冠状病毒感染主要导致宿主呼吸系统、肝脏、胃肠道以及神经系统出现急性或慢性症状。2000年以来,传染性非典型肺炎和中东呼吸综合征的暴发,以及猪流行性腹泻病毒在全球猪群中的暴发流行,引起大家对动物冠状病毒的极大重视。S蛋白具有受体结合活性和膜融合活性,是冠状病毒感染细胞的关键蛋白;S蛋白在病毒的组织或宿主嗜性和毒力等方面发挥重要作用。本文重点对近年来冠状病毒S蛋白的结构、功能以及S蛋白与受体相互作用的研究进行综述,以期为冠状病毒的入侵机制和反向遗传学研究以及受体阻断药物的开发提供参考。     

Review: Microbial endocrinology: intersection of microbiology and neurobiology matters to swine health from infection to behavior

From birth to slaughter, pigs are in constant interaction with microorganisms. Exposure of the skin, gastrointestinal and respiratory tracts, and other systems allows microorganisms to affect the developmental trajectory and function of porcine physiology as well as impact behavior. These routes of communication are bi-directional, allowing the swine host to likewise influence microbial survival, function and community composition. Microbial endocrinology is the study of the bi-directional dialogue between host and microbe. Indeed, the landmark discovery of host neuroendocrine systems as hubs of host–microbe communication revealed neurochemicals act as an inter-kingdom evolutionary-based language between microorganism and host. Several such neurochemicals are stress catecholamines, which have been shown to drastically increase host susceptibility to infection and augment virulence of important swine pathogens, including Clostridium perfringens. Catecholamines, the production of which increase in response to stress, reach the epithelium of multiple tissues, including the gastrointestinal tract and lung, where they initiate diverse responses by members of the microbiome as well as transient microorganisms, including pathogens and opportunistic pathogens. Multiple laboratories have confirmed the evolutionary role of microbial endocrinology in infectious disease pathogenesis extending from animals to even plants. More recent investigations have now shown that microbial endocrinology also plays a role in animal behavior through the microbiota–gut–brain axis. As stress and disease are ever-present, intersecting concerns during each stage of swine production, novel strategies utilizing a microbial endocrinology-based approach will likely prove invaluable to the swine industry.     

The influence of host demography, pathogen virulence, and relationships with pathogen virulence on the evolution of pathogen transmission in a spatial context

A major challenge in evolutionary ecology is to explain extensive natural variation in transmission rates and virulence across pathogens. Host and pathogen ecology is a potentially important source of that variation. Theory of its effects has been developed through the study of non-spatial models, but host population spatial structure has been shown to influence evolutionary outcomes. To date, the effects of basic host and pathogen demography on pathogen evolution have not been thoroughly explored in a spatial context. Here we use simulations to show that space produces novel predictions of the influence of the shape of the pathogen’s transmission–virulence tradeoff, as well as host reproduction and mortality, on the pathogen’s evolutionary stable transmission rate. Importantly, non-spatial models predict that neither the slope of linear transmission–virulence relationships, nor the host reproduction rate will influence pathogen evolution, and that host mortality will only influence it when there is a transmission–virulence tradeoff. We show that this is not the case in a spatial context, and identify the ecological conditions under which spatial effects are most influential. Thus, these results may help explain observed natural variation among pathogens unexplainable by non-spatial models, and provide guidance about when space should be considered. We additionally evaluate the ability of existing analytical approaches to predict the influence of ecology, namely spatial moment equations closed with an improved pair approximation (IPA). The IPA is known to have limited accuracy, but here we show that in the context of pathogens the limitations are substantial: in many cases, IPA incorrectly predicts evolution to pathogen-driven extinction. Despite these limitations, we suggest that the impact of ecology can still be understood within the conceptual framework arising from spatial moment equations, that of “self-shading’’, whereby the spread of highly transmissible pathogens is impeded by local depletion of susceptible hosts.     

IS HIV SHORT‐SIGHTED? INSIGHTS FROM A MULTISTRAIN NESTED MODEL

An important component of pathogen evolution at the population level is evolution within hosts. Unless evolution within hosts is very slow compared to the duration of infection, the composition of pathogen genotypes within a host is likely to change during the course of an infection, thus altering the composition of genotypes available for transmission as infection progresses. We develop a nested modeling approach that allows us to follow the evolution of pathogens at the epidemiological level by explicitly considering within‐host evolutionary dynamics of multiple competing strains and the timing of transmission. We use the framework to investigate the impact of short‐sighted within‐host evolution on the evolution of virulence of human immunodeficiency virus (HIV), and find that the topology of the within‐host adaptive landscape determines how virulence evolves at the epidemiological level. If viral reproduction rates increase significantly during the course of infection, the viral population will evolve a high level of virulence even though this will reduce the transmission potential of the virus. However, if reproduction rates increase more modestly, as data suggest, our model predicts that HIV virulence will be only marginally higher than the level that maximizes the transmission potential of the virus.     

Vascular Endothelial Growth Factor A Promotes Vaccinia Virus Entry into Host Cells via Activation of the Akt Pathway

Vaccinia virus (VV) is an enveloped DNA virus from the poxvirus family and has played a crucial role in the eradication of smallpox. It continues to be used in immunotherapy for the prevention of infectious diseases and treatment of cancer. However, the mechanisms of poxvirus entry, the host factors that affect viral virulence, and the reasons for its natural tropism for tumor cells are incompletely understood. By studying the effect of hypoxia on VV infection, we found that vascular endothelial growth factor A (VEGF-A) augments oncolytic VV cytotoxicity. VEGF derived from tumor cells acts to increase VV internalization, resulting in increased replication and cytotoxicity in an AKT-dependent manner in both tumor cells and normal respiratory epithelial cells. Overexpression of VEGF also enhances VV infection within tumor tissue in vivo after systemic delivery. These results highlight the importance of VEGF expression in VV infection and have potential implications for the design of new strategies to prevent poxvirus infection and the development of future generations of oncolytic VV in combination with conventional or biological therapies.     

The Double-Edged Sword: How Evolution Can Make or Break a Live-Attenuated Virus Vaccine

Even students who reject evolution are often willing to consider cases in which evolutionary biology contributes to, or undermines, biomedical interventions. Moreover, the intersection of evolutionary biology and biomedicine is fascinating in its own right. This review offers an overview of the ways in which evolution has impacted the design and deployment of live-attenuated virus vaccines, with subsections that may be useful as lecture material or as the basis for case studies in classes at a variety of levels. Live-attenuated virus vaccines have been modified in ways that restrain their replication in a host so that infection (vaccination) produces immunity but not disease. Applied evolution, in the form of serial passage in novel host cells, is a “classical” method to generate live-attenuated viruses. However, many live-attenuated vaccines exhibit reversion to virulence through back-mutation of attenuating mutations, compensatory mutations elsewhere in the genome, recombination or reassortment, or changes in quasispecies diversity. Additionally, the combination of multiple live-attenuated strains may result in competition or facilitation between individual vaccine viruses, resulting in undesirable increases in virulence or decreases in immunogenicity. Genetic engineering informed by evolutionary thinking has led to a number of novel approaches to generate live-attenuated virus vaccines that contain substantial safeguards against reversion to virulence and that ameliorate interference among multiple vaccine strains. Finally, vaccines have the potential to shape the evolution of their wild-type counterparts in counter-productive ways; at the extreme, vaccine-driven eradication of a virus may create an empty niche that promotes the emergence of new viral pathogens.     

Imperfect vaccines and the evolution of pathogens causing acute infections in vertebrates

A study by Gandon et al. (2001) considered the potential ways pathogens may evolve in response to vaccination with imperfect vaccines. In this paper, by focusing on acute infections of vertebrate hosts, we examine whether imperfect vaccines that do not completely block a pathogen's replication (antigrowth) or transmission (antitransmission) may lead to evolution of more or less virulent pathogen strains. To address this question, we use models of the within-host dynamics of the pathogen and the host's immune responses. One advantage of the use of this within-host approach is that vaccination can be easily incorporated in the models and the trade-offs between pathogen transmissibility, host recovery, and virulence that drive evolution of pathogens in these models can be easily estimated. We find that the use of either antigrowth or antitransmission vaccines leads to the evolution of pathogens with an increased within-host growth rate; infection of unvaccinated hosts with such evolved pathogens results in high host mortality and low pathogen transmission. Vaccination of only a fraction of hosts with antigrowth vaccines may prevent pathogens from evolving high virulence due to pathogen adaptation to unvaccinated hosts and thus protection of vaccinated hosts from pathogen-induced disease. In contrast, antitransmission vaccines may be beneficial only if they are effective enough to cause pathogen extinction. Our results suggest that particular mechanisms of action of vaccines and their efficacy are crucial in predicting longterm evolutionary consequences of the use of imperfect vaccines.     

Strain variation in an emerging iridovirus of warm-water fishes

Although iridoviruses vary widely within and among genera with respect to their host range and virulence, variation within iridovirus species has been less extensively characterized. This study explores the nature and extent of intraspecific variation within an emerging iridovirus of North American warm-water fishes, largemouth bass virus (LMBV). Three LMBV isolates recovered from three distinct sources differed genetically and phenotypically. Genetically, the isolates differed in the banding patterns generated from amplified fragment length polymorphism analysis but not in their DNA sequences at two loci of different degrees of evolutionary stability. In vitro, the isolates replicated at identical rates in cell culture, as determined by real-time quantitative PCR of viral particles released into suspension. In vivo, the isolates varied over fivefold in virulence, as measured by the rate at which they induced mortality in juvenile largemouth bass. This variation was reflected in the viral loads of exposed fish, measured using real-time quantitative PCR; the most virulent viral strain also replicated to the highest level in fish. Together, these results justify the designation of these isolates as different strains of LMBV. Strain variation in iridoviruses could help explain why animal populations naturally infected with iridovirus pathogens vary so extensively in their clinical responses to infection. The results of this study are especially relevant to emerging iridoviruses of aquaculture systems and wildlife.     

Electrostatic modeling predicts the activities of orthopoxvirus complement control proteins

Regulation of complement activation by pathogens and the host are critical for survival. Using two highly related orthopoxvirus proteins, the vaccinia and variola (smallpox) virus complement control proteins, which differ by only 11 aa, but differ 1000-fold in their ability to regulate complement activation, we investigated the role of electrostatic potential in predicting functional activity. Electrostatic modeling of the two proteins predicted that altering the vaccinia virus protein to contain the amino acids present in the second short consensus repeat domain of the smallpox protein would result in a vaccinia virus protein with increased complement regulatory activity. Mutagenesis of the vaccinia virus protein confirmed that changing the electrostatic potential of specific regions of the molecule influences its activity and identifies critical residues that result in enhanced function as measured by binding to C3b, inhibition of the alternative pathway of complement activation, and cofactor activity. In addition, we also demonstrate that despite the enhanced activity of the variola virus protein, its cofactor activity in the factor I-mediated degradation of C3b does not result in the cleavage of the alpha' chain of C3b between residues 954-955. Our data have important implications in our understanding of how regulators of complement activation interact with complement, the regulation of the innate immune system, and the rational design of potent complement inhibitors that might be used as therapeutic agents.     

An Outer Membrane Receptor of Neisseria meningitidis Involved in Zinc Acquisition with Vaccine Potential

Since the concentration of free iron in the human host is low, efficient iron-acquisition mechanisms constitute important virulence factors for pathogenic bacteria. In Gram-negative bacteria, TonB-dependent outer membrane receptors are implicated in iron acquisition. It is far less clear how other metals that are also scarce in the human host are transported across the bacterial outer membrane. With the aim of identifying novel vaccine candidates, we characterized in this study a hitherto unknown receptor in Neisseria meningitidis. We demonstrate that this receptor, designated ZnuD, is produced under zinc limitation and that it is involved in the uptake of zinc. Upon immunization of mice, it was capable of inducing bactericidal antibodies and we could detect ZnuD-specific antibodies in human convalescent patient sera. ZnuD is highly conserved among N. meningitidis isolates and homologues of the protein are found in many other Gram-negative pathogens, particularly in those residing in the respiratory tract. We conclude that ZnuD constitutes a promising candidate for the development of a vaccine against meningococcal disease for which no effective universal vaccine is available. Furthermore, the results suggest that receptor-mediated zinc uptake represents a novel virulence mechanism that is particularly important for bacterial survival in the respiratory tract.     

Phage-Driven Loss of Virulence in a Fish Pathogenic Bacterium

Parasites provide a selective pressure during the evolution of their hosts, and mediate a range of effects on ecological communities. Due to their short generation time, host-parasite interactions may also drive the virulence of opportunistic bacteria. This is especially relevant in systems where high densities of hosts and parasites on different trophic levels (e.g. vertebrate hosts, their bacterial pathogens, and virus parasitizing bacteria) co-exist. In farmed salmonid fingerlings, Flavobacterium columnare is an emerging pathogen, and phage that infect F. columnare have been isolated. However, the impact of these phage on their host bacterium is not well understood. To study this, four strains of F. columnare were exposed to three isolates of lytic phage and the development of phage resistance and changes in colony morphology were monitored. Using zebrafish (Danio rerio) as a model system, the ancestral rhizoid morphotypes were associated with a 25–100% mortality rate, whereas phage-resistant rough morphotypes that lost their virulence and gliding motility (which are key characteristics of the ancestral types), did not affect zebrafish survival. Both morphotypes maintained their colony morphologies over ten serial passages in liquid culture, except for the low-virulence strain, Os06, which changed morphology with each passage. To our knowledge, this is the first report of the effects of phage-host interactions in a commercially important fish pathogen where phage resistance directly correlates with a decline in bacterial virulence. These results suggest that phage can cause phenotypic changes in F. columnare outside the fish host, and antagonistic interactions between bacterial pathogens and their parasitic phage can favor low bacterial virulence under natural conditions. Furthermore, these results suggest that phage-based therapies can provide a disease management strategy for columnaris disease in aquaculture.     

Naturally Occurring Swine Influenza A Virus PB1-F2 Phenotypes That Contribute to Superinfection with Gram-Positive Respiratory Pathogens

A combination of viral, bacterial, and host factors contributes to the severity and overall mortality associated with influenza virus-bacterium superinfections. To date, the virulence associated with the recently identified influenza virus protein PB1-F2 has been largely defined using models of primary influenza virus infection, with only limited assessment in models of Streptococcus pneumoniae superinfection. Specifically, these studies have incorporated isogenic viruses that differ in the PB1-F2 expressed, but there is still knowledge to be gained from evaluation of natural variants derived from a nonhuman host species (swine). Using this rationale, we developed the hypothesis that naturally occurring viruses expressing variants of genes, like the PB1-F2 gene, can be associated with the severity of secondary bacterial infections. To test this hypothesis, we selected viruses expressing variants in PB1-F2 and evaluated outcomes from superinfection with three distinct Gram-positive respiratory pathogens: Streptococcus pneumoniae, Staphylococcus aureus, and Streptococcus pyogenes. Our results demonstrate that the amino acid residues 62L, 66S, 75R, 79R, and 82L, previously proposed as molecular signatures of PB1-F2 virulence for influenza viruses in the setting of bacterial superinfection, are broadly associated with enhanced pathogenicity in swine in a bacterium-specific manner. Furthermore, truncated PB1-F2 proteins can preferentially increase mortality when associated with Streptococcus pyogenes superinfection. These findings support efforts to increase influenza virus surveillance to consider viral genotypes that could be used to predict increased severity of superinfections with specific Gram-positive respiratory pathogens.